One of the most exciting areas in biology is the nervous system and how it works. Viral infections of the nervous system have provided exceptional insight at many levels, from pathogenesis to basic biology. The mechanism(s) by which alpha herpesvirus infections spread into, within, and out of the nervous system are understood in principle, but not in any detail despite considerable effort. This unique biology leads to efficient host-to-host transmission and establishment of these viruses in their natural host populations with minimal pathogenesis. A long-term goal of my laboratory is to determine the molecular mechanisms by which neuroinvasive alpha-herpesviruses move in and out of the mammalian nervous system. These mechanisms will provide targets for manipulation that could substantially expand our understanding of infection transmission. Work in this renewal proposal continues to build on powerful imaging technology developed in the past funding period to reveal how herpes virion components move inside neurons and from neurons to non-neuronal cells in vitro and in vivo. We seek to identify and quantify critical events and potential bottlenecks in long distance transmission of infection from the peripheral nervous system (PNS) to peripheral epithelial cells. Experiments are divided among three aims all featuring light and video microscopy: Imaging individual virion egress events using multi-color TIRF microscopy; assaying axon-cell egress and spread events with chambered neurons, three color virus technology, and fast epifluorescence imaging; and imaging in vivo/ex vivo PRV invasion of the PNS at the single cell and single particle level. The technology and knowledge obtained from these studies have broad application. They enable a better understanding of herpesvirus cell biology at the single cell and particle level, provide insight into potential bottlenecks during hot-host transmission and have implications for intervention strategies. This technology and knowledge also will provide opportunities to develop enhanced viral tracers for understanding the organization and functional architecture of the nervous system.